Apparatus and method for determination of the adequacy of dialysis by non-invasive near-infrared spectroscopy

ABSTRACT

Methods and apparatus for non-invasive tissue urea concentrations during or subsequent to hemodialysis using near-infrared spectroscopy are discussed. Near-infrared tissue spectra can be obtained by projecting near-infrared radiation into skin on the underside of human forearms and capturing the light reflected back and out through the tissue. An index matching medium is used to couple the tissue to the analyzer. The tissue spectrum collected preferably includes primarily diffuse reflected light reflected from the inner dermis. Multiple tissue spectra of known urea concentration are used to build a model from which the urea concentration of an unknown sample can be devised. The model is based on a partial least squares algorithm applied to multiple tissue scans and concomitant blood sample urea measurements. This model is then applied to an unknown tissue spectra.

CROSS REFERENCE TO RELATED PATENTS AND PENDING APPLICATIONS

The present application is related to U.S. patent application Ser. No.09/174,812, filed Oct. 19, 1998, entitled “Method for Non-InvasiveAnalyte Measurement with Improved Optical Interface”; U.S. patentapplication Ser. No. 08/871,366, filed Jun. 9, 1997, entitled “DiffuseReflectance Monitoring Apparatus”; and U.S. patent application Ser. No.08/961,323, filed Oct. 30, 1997, entitled “Dialysis Monitoring Methodand Apparatus”, all of which are assigned to the same assignee as thepresent application.

TECHNICAL FIELD

The present invention relates generally to methods and systems fordetermining the adequacy of treatment during hemodialysis of a patientutilizing a non-invasive near-infrared tissue analysis. Morespecifically, the invention relates to direct measurement of ureaconcentrations in tissue of patients undergoing dialysis with lightdiffusely reflected by skin in conjunction with a spectrographic model,which relates urea concentration to a diffusely reflected lightspectrum.

BACKGROUND OF THE INVENTION

Measurement of the efficacy of hemodialysis treatments is currently timeconsuming, inaccurate and expensive. Approximately 260,000 Americanssuffer from end-stage renal disease (ESRD). Fifty-nine percent aretreated by thrice-weekly maintenance hemodialysis sessions designed toclear the products of metabolism that are normally excreted by thekidneys in the urine. Since the failure to adequately dialyze a patienthas been shown to increase mortality and morbidity and since the processof dialyzing an ESRD patient is complex and variable in terms of theefficiency of the treatment, a number of methods have been developed toquantify the effectiveness of the treatment. The technique used in theoverwhelming majority of dialysis centers is based on pre- andpost-dialysis measurements of blood urea nitrogen concentrations. Urea,a low-molecular weight molecule, is a product of protein metabolism thatis normally cleared from the body by the kidneys. Because it is alsocleared from the blood by the dialysis process and easily measured inblood, its disappearance from the blood during hemodialysis is a measureof the efficacy or adequacy of that particular treatment session. Theprocess of removal of toxins from the body by hemodialysis is bestrepresented as a logarithmic function. As such, the coefficient of thenatural logarithm termed KT/Vd, which is calculated from pre- andpost-dialysis measurements of blood urea concentrations, can be used asa single descriptor of dialysis adequacy.

The importance of adequate duration or dose of hemodialysis has beenunderscored recently by the observation that the adjusted mortality ofpatients with renal disease in the United States exceeds that of severalother countries, despite a longer life expectancy of the generalpopulation of the United States. A number of studies have documented thefailure to deliver an adequate dose of hemodialysis to many Americans.The failure of delivery of adequate hemodialysis doses in the UnitedStates is a result of many factors. Time and financial pressurescontribute to the problem. Because the metabolic toxins are removed fromthe blood, which makes up only a fraction of the total volume of thebody in which the toxins are distributed, there are delays as thesolutes redistribute and equilibrate after dialysis. Thus, measurementof KT/Vd is highly dependent on the time of the urea measurements andthe relative size of the compartments such as blood water, interstitialwater and intracellular water, all of which harbor urea and othercontaminants. These compartments vary in size from patient to patient,and within a patient depending upon present physiologic state. The bestmeasure of the post-dialysis urea is made at least 15 minutes afterhemodialysis, but for some patients it may require 50 to 60 minutes toreach equilibrium. There is no accurate way to predict which patientswill have a significant blood urea increase following hemodialysis atany given treatment time. Given the time constraints on out-patienthemodialysis centers that commonly are able to dialyze no more than twopatients per day on a single machine, one in the morning and one in theafternoon, the need to obtain post-dialysis blood urea concentrations 30to 60 minutes after dialysis is impractical at best. Finally, the lateblood measurement requires an additional venipuncture of the patient whois disconnected from the dialysis machine minutes after cessation ofcirculation through the machine.

Urea testing is a capital burden on the dialysis centers that providedialysis to ESRD patients under a capitated reimbursement basis. Theblood drawing process is labor intensive and exposes the nursing staffto blood borne pathogens. The samples must then be transported to alaboratory for analysis, incurring another charge and a delay inreported values. Currently, the accepted “standard of care” givenfinancial constraints is that KT/Vd be measured once per month, that is,once during every 12 dialysis sessions. In summary, hemodialysis is“under-delivered” in the United States. Financial and time constraintsresult in failure to recognize such inadequacy given the infrequentcollection of blood for urea samples and calculation of KT/Vd, as wellas poor modeling due to variability of the rebound effect and early postdialysis blood collection.

As noted above, monitoring the adequacy of hemodialysis, as defined bythe National Kidney Foundation (NKF)—“1997 DOQI Clinical PracticeGuidelines for Hemodialysis Adequacy”, and the Renal PhysiciansAssociations (RPA) “1993 Clinical Practice Guidelines on Adequacy ofHemodialysis”, entail measuring blood urea nitrogen (BUN) pre- andpost-dialysis once per month in order to calculate the so-called singlepool KT/Vd value with K=dialyzer clearance, T=time of dialysis, andV=volume of distribution of urea. KT/Vd is then calculated from pre- andpost-dialysis BUN concentrations by the following formula:

KT/V=−Ln(Ct/Co−0.008t−UF/W)

Where Ct is the post-dialysis urea level and Co is the pre-dialysis urealevel; t is the time; UF is the ultrafiltrate removed; and W is thepost-dialysis weight.

The equation is most representative of the true dialysis dose if thepost session BUN blood sample is drawn after the blood urea hasequilibrated with the interstitial and intracellular urea. Release ofsequestered urea from the intracellular space to the extracellular spacecontinues for 30 to 60 minutes after completion of a dialysis session.This equilibration is due to the removal of urea from the blood by thedialyzer at a rate that exceeds the rate of diffusion from theintracellular to the extracellular compartment. Delays of equilibriumare also caused by the so-called “flow-volume disequilibrium”. Seventypercent of the total body water is contained in organs that receive only20% of the cardiac output. Relatively poorly perfused tissues such asskin, muscle, and bone are cleared of urea less efficiently than highlyvascularized organs such as the liver or lungs. The consequence of thiscompartmentalization of urea is an increase in the BUN concentrationover the 60 minutes after the completion of hemodialysis.

The magnitude of the urea rebound varies greatly among dialysispatients. The average increase of urea concentration in the 30 minutesfollowing completion of dialysis is 17%. However, some patients exhibita rebound as high as 45%. This results in a 75% error between KT/V basedon immediate post-dialysis BUN measurement and 30 minutes post-dialysisdetermination. Despite these limitations of the single-pool KT/V modelbased on the immediate post-dialysis BUN, the need to obtain thepost-dialysis BUN sample 30 to 60 minutes after the completion ofdialysis in order to compute the more accurate double-pool KT/V, isimpractical in the out-patient hemodialysis setting.

At least two methods for approximating the equilibrated or double-poolKT/Vd have been proposed in the literature. The Smye formulaapproximates the equilibration BUN concentration based on three ureameasurements, the usual pre- and post-dialysis determinations, as wellas a mid-dialysis blood sample. This method yields an average error of13% between the estimated equilibration KT/V and the true equilibratedvalue. The Daugirdas formulas are based on linear transformations of thesingle-pool KT/V modified according to the type of vascular access;venous shunt or arterial shunt. The improvement of accuracy iscomparable to the Smye method.

Despite limitations of the single pool technique, the NKF recommends itsuse because of the impracticality of the late measurement of urea in theout-patient setting and the unproven accuracy of the double poolestimates.

Living human tissue and blood is recognized as a dynamic systemcontaining a multitude of components and analyte information that isparticularly useful in the medical profession for diagnosing, treatingand monitoring human physical conditions. To this end, effort has beendirected toward developing methods for non-invasive measurement oftissue and blood constituents using spectroscopy. The spectrographicanalysis of living tissue has been focused on the identification ofspectral information that defines individual analytes and relating suchspectral data to the analyte's concentration. Concentration of theseanalytes vary with time in an individual patient. Acquiring tissuespectral data with sufficient accuracy for use in diagnosis andtreatment has proven difficult. Difficulties in conducting the analysishave been found, which are related to the fact that the tissue system isa complex matrix of materials with differing refractive indices andabsorption properties. Further, because the constituents of interest aremany times present at very low concentrations, high concentrationconstituents, such as water, have had a detrimental impact onidentifying the low level constituent spectral information and giving anaccurate reading of the desired constituent concentration.

Improved methods and apparatus for gathering and analyzing anear-infrared tissue spectra for an analyte concentration are disclosedin commonly assigned U.S. Patent applications and issued patents. U.S.Pat. No. 5,655,530 and U.S. patent application Ser. No. 08/844,501,filed Apr. 18, 1997, entitled “Method for Non-invasive Blood AnalyteMeasurement with Improved Optical Interface” relate to near-infraredanalysis of a tissue analyte concentration which varies with time, witha primary focus on glucose concentrations in diabetic individuals. Themethods and apparatus include placing a refractive index-matching mediumbetween a sensor and the skin to improve the accuracy and repeatabilityof testing. U.S. patent application Ser. No. 09/174,812, filed Oct. 19,1998, entitled “Method for Non-Invasive Blood Analyte Measurement withImproved Optical Interface” discloses additional improvements innon-invasive living tissue analyte analysis. The disclosure of each ofthese three applications or patents are hereby incorporated byreference.

U.S. Pat. No. 5,636,633 relates, in part, to another aspect of accuratenon-invasive measurement of an analyte concentration. The apparatusincludes a device having transparent and reflective quadrants forseparating diffuse reflected light from specular reflected light.Incident light projected into the skin results in specular and diffusereflected light coming back from the skin. Specular reflected light haslittle or no useful information and is preferably removed prior tocollection. U.S. patent application Ser. No. 08/871,366, filed Jun. 9,1997, entitled “Improved Diffuse Reflectance Monitoring Apparatus”,discloses a further improvement for accurate analyte concentrationanalysis which includes a blocking blade device for separating diffusereflected light from specular reflected light. The blade allows lightfrom the deeper, inner dermis layer to be captured, rejecting light fromthe surface, epidermis layer, where the epidermis layer has much lessanalyte information than the inner dermis layer, and contributes noise.The blade traps specular reflections as well as diffuse reflections fromthe epidermis. The disclosures of the above patent and application,which are assigned to the assignee of the present application, are alsoincorporated herein by reference.

SUMMARY OF THE INVENTION

The present invention is directed to a method and apparatus to directlymeasure the urea concentration of patients undergoing dialysis, usingreflectance, non-invasive near-infrared (NIR) spectroscopy. Thus,instead of drawing blood samples from the patient at the beginning andend of dialysis for analysis of BUN concentrations at a clinicallaboratory, the patient's skin will be “scanned” and BUN concentrationdetermined in real-time. This will allow the calculation of dialysisdose or KT/Vd to be measured and reported by the end of the dialysissession.

In its broadest sense, the present invention includes a method forassessing the need for hemodialysis, the progress of a hemodialysisprocedure or the adequacy of a hemodialysis treatment. The methodgenerally includes providing a means for optical analysis of tissue on apatient with the means for optical analysis providing an output spectrumat multiple wavelengths. Tissue, as defined herein, includes all tissuecomponents found in a given cross section that is optically penetratedduring analysis. The tissue is chiefly made up of extravascular water,which includes both interstitial and intracellular fluid, with arelatively small fraction comprising blood. The output spectrum hasvarying intensities as related to absorption by the non-vascular tissue.The tissue is coupled to the means for optical analysis, and an outputspectrum is acquired before, during or after hemodialysis. The tissueurea concentration, in contrast to blood urea concentration, iscalculated from a mathematical model relating the output spectrum to thetissue urea concentration.

In the most limited case, the method and apparatus of the presentinvention would be used in dialysis centers to measure the pre- andpost-dialysis urea levels of patients for real-time KT/V calculations.In a preferred method, measurement can also be made continuously formore detailed urea removal modeling or for feedback to the dialysis unitfor feedback and control purposes.

The advantages of the method of the present invention over the standardmethod of calculation of KT/V by pre- and post-dialysis blood sample BUNmeasurement are four-fold. First, the accuracy of the calculation ofKT/V measured at the end of dialysis approaches the accuracy of KT/Vbased on post equilibrium blood sample BUN measurements. This is due tothe fact that the analysis is of tissue versus blood. The tissue has arelatively small (and negative) urea rebound. Because the tissue wateris both chiefly extravascular and much of it is relatively poorlyvascularized, its urea content more closely correlates with equilibratedtotal body urea and equilibrated blood sample BUN than does theimmediate post-dialysis, pre-rebound blood sample BUN. Thus, thenon-invasive skin measurement of urea delivers the accuracy of theequilibrated or two-pool KT/V, but does not require that the dialysispatient and staff wait 30 minutes or more after dialysis beforecollecting a final blood sample.

Second, calculation of KT/V is nearly “real-time”. The clinician orstaff overseeing the dialysis session will be able to judge the efficacyor adequacy of the dialysis dose at the time it is delivered in a “pointof care” mode. Failure to deliver the prescribed dose can be appreciatedbefore the patient leaves the dialysis clinic. A decision can then bemade to continue the current session or adjust the following dialysissession dose. Should the machine be devoted to a single patient duringdialysis, true, real-time kinetic modeling is possible.

Third, the non-invasive nature of the measurement limits the exposure ofnursing and technical staff to blood born infectious agents. Two bloodsamples must be removed from the closed loop hemodialysis circuit andtransferred to standard blood analysis tubes in order to calculate theKT/V. In an average hemodialysis centers, where 100 clients aredialyzed, at least 200 blood samples are drawn and sent via courier to aclinical chemistry laboratory. Replacement of blood sample BUNmeasurements by non-invasive skin measurements would substantiallyreduce the potential of infection of both the nursing staff who drawblood samples and laboratory personnel who then handle the specimens.

Finally, a fourth advantage over the current methods is a reduction ofthe cost per measurement. Although the investment in such a device willhave to be considered in terms of the fixed reimbursement for monthlymeasures of KT/V, the clinician will not incur significant costs byusing the device more often than once per month. In fact, like the usualblood pressure measurement at the end of a dialysis session, anon-invasive measure of urea would require no reoccurring costs exceptthe time to make the measurement. Dialysis patients would greatlybenefit from more frequent measurement of the dialysis dose.

Success of the method of the present invention is believed tied to twocomponents. First, the method incorporates an apparatus and techniquefor accurately and repeatably acquiring a tissue spectra which is bothstable and sensitive to slight changes in spectral output at desiredwavelengths and optimizes optical throughput both into and out of thetissue sample. Second, because the spectral features, which can becorrelated to tissue urea concentration are not readily apparent fromthe spectral data, a mathematical model is utilized to correlatespectral data to a tissue urea concentration. The model is built basedon multiple tissue scans and same time blood sample BUN measurements.The method preferably incorporates a resultant mathematical model basedon Partial Least Squares Algorithm applied to the multiple tissue scansand concomitant BUN measurements which is then applied to an unknownspectra.

The present invention, thus, includes a method for measuring tissue ureaconcentration of an individual before, during or just after hemodialysisusing non-invasive tissue spectroscopy. A preferred method and apparatusilluminates skin with near-infrared radiation and collects thereflected, non-absorbed near-infrared radiation. Diffuse, rather thanspecular, reflected light is preferably collected, more preferably lightdiffusely reflected from the inner dermis rather than the epidermis. Thenear-infrared spectra collected can be stored in a computer database.

The method for non-invasively measuring the concentration of urea intissue includes first providing an apparatus for measuring infraredabsorption by a urea containing tissue. The apparatus includes generallythree elements, an energy source, a sensor element, and a spectrumanalyzer. The sensor element includes an input element and an outputelement. The input element is operatively connected to the energy sourceby a first means for transmitting infrared energy. The output element isoperatively connected to the spectrum analyzer by a second means fortransmitting infrared energy.

In preferred embodiments, the input element and output element compriselens systems which focus the infrared light energy to and from thesample. In a preferred embodiment, the input element and output elementcomprise a single lens system which is utilized for both input ofinfrared light energy from the energy source and output of both specularand diffusely reflected light energy from the analyte-containing sample.Alternatively, the input element and output element can comprise twolens systems, placed on opposing sides of an analyte-containing sample,wherein light energy from the energy source is transmitted to the inputelement and light energy transmitted through the urea-containing samplethen passes through the output element to the spectrum analyzer.

The first means for transmitting infrared energy, in preferredembodiments, simply includes placing the infrared energy sourceproximate to the input element so that light energy from the source istransmitted via the air to the input element. Further, in preferredembodiments, the second means for transmitting infrared energypreferably includes a single mirror or system of mirrors which directthe light energy exiting the output element through the air to thespectrum analyzer.

In practicing the method of the present invention, a urea-containingtissue area is selected as the point of analysis. This area can includethe skin surface on the finger, earlobe, forearm or any other skinsurface. Preferably, the urea-containing tissue is the underside of theforearm.

A quantity of an index-matching medium or fluid is then placed on theskin area to be analyzed. The index-matching fluid detailed herein isselected to optimize introduction of light into the tissue, reducespecular light and effectively get light out of the tissue. The mediumor fluid preferably contains an additive which confirm proper couplingto the skin surface by a proper fluid, thus assuring the integrity oftest data. It is preferred that the index-matching medium is non-toxicand has a spectral signature in the near-infrared region which isminimal, and is thus minimally absorbing of light energy havingwavelengths relevant to the urea being measured. In preferredembodiments, the index-matching medium has a refractive index of about1.38. Further, the refractive index of the medium is preferably constantthroughout the composition.

The sensor element, which includes the input element and the outputelement, is then placed in contact with the index-matching medium. Inthis way, the input element and output element are coupled to theurea-containing tissue or skin surface via the index-matching mediumwhich eliminates the need for the light energy to propagate through airor pockets of air due to irregularities in the skin surface.

In analyzing for the concentration of urea in the tissue, light energyfrom the energy source is transmitted via the first means fortransmitting infrared energy into the input element. The light energy istransmitted from the input element through the index-matching medium tothe skin surface. Some of the light energy contacting theanalyte-containing sample is differentially absorbed by the variouscomponents and analytes contained therein at various depths within thesample. Some of the light energy is also transmitted through the sample.However, a quantity of light energy is reflected back to the outputelement. In a preferred embodiment, the non-absorbed or non-transmittedlight energy is reflected back to the output element upon propagatingthrough the index-matching medium. This reflected light energy includesboth diffusely reflected light energy and specularly reflected lightenergy. Specularly reflected light energy is that which reflects fromthe surface of the sample and contains little or no analyte information,while diffusely reflected light energy is that which reflects fromdeeper within the sample, wherein the analytes are present.

In preferred embodiments, the specularly reflected light energy isseparated from the diffusely reflected light energy. The non-absorbeddiffusely reflected light energy is then transmitted via the secondmeans for transmitting infrared energy to the spectrum analyzer. Asdetailed below, the spectrum analyzer preferably utilizes a computer togenerate a urea concentration utilizing the measured intensities, acalibration model, and a multivariate algorithm.

The computer includes a memory having stored therein a multivariatecalibration model empirically relating the known urea concentration in aset of calibration samples to the measure intensity variations from thecalibration samples, at several wavelengths.

Such a model is constructed using techniques known by statisticians.

The computer predicts the analyte con centration of the urea-containingsample by utilizing the measure intensity variations, calibration modeland a multivariate algorithm. Preferably, the computation is made by thepartial least square technique as disclosed by Robinson et al. in U.S.Pat. No. 4,975,581, incorporated herein by reference.

These and various other advantages and features of novelty whichcharacterize the present invention are pointed out with particularity inthe claims annexed hereto and forming a part hereof. However, for abetter understanding of the invention, its advantages, and the objectobtained by its use, reference should be made to the drawings which forma further part hereof, and to the accompanying descriptive matter inwhich there are illustrated and described preferred embodiments of thepresent invention.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, in which like reference numerals indicate correspondingparts or elements of preferred embodiments of the present inventionthroughout the several views:

FIG. 1 is a graphical representation of blood urea versus tissue ureaconcentrations during dialysis and illustrating the rebound effect;

FIG. 2 is a partial cross-sectional view of a sensor element coupled tothe skin surface via an indexing-matching fluid;

FIG. 3 is a partial cross-sectional view of an alternative embodiment ofa sensor element coupled to opposite sides of a skin surface via anindexing-matching fluid;

FIG. 4 is a graphical representation of experimental data showing theimprovement in accuracy and repeatability of a sensor coupled to theskin via an index-matching medium;

FIG. 5 is a graphical representation of a single patient's experimentaldata showing blood urea plotted relative to tissue urea predicted by themethods of the present invention;

FIG. 6 is a second graphical representation of a second patient's bloodurea plotted relative to predicted tissue urea utilizing the methods ofthe present invention;

FIG. 7 is a graph showing the blood urea plotted against the tissue ureafor the data of FIG. 5; and

FIG. 8 is a graph showing the blood urea plotted against the tissue ureafor the data of FIG. 6.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Detailed embodiments of the present invention are disclosed herein.However, it is to be understood that the disclosed embodiments aremerely exemplary of the present invention which may be embodied invarious systems. Therefore, specific details disclosed herein are not tobe interpreted as limiting, but rather as a basis for the claims and asa representative basis for teaching one of skill in the art to variouslypractice the invention.

The present invention is directed to an apparatus and method fordirectly measuring the urea concentration in tissue of patientsundergoing or just completing dialysis. The method utilizes reflectance,non-invasive near-infrared spectroscopy. Thus, instead of drawing bloodsamples from a patient at the beginning and end of dialysis for analysisof blood sample urea concentrations at a clinical laboratory, thepatient's skin is scanned and urea concentrations determined in realtime.

The present invention is directed toward a method and apparatus whichovercomes the deficiencies in present dialysis related to blood urearebound subsequent to termination of a dialysis procedure. The reboundis caused by the fact that a blood sample is drawn at a time when theblood urea has not equilibrated with the interstitial and intracellularurea. This phenomena is depicted graphically in FIG. 1 and referenceshould be made thereto. Release of sequestered urea from theintracellular space to the extracellular space continues for about 30 toabout 60 minutes after completion of a dialysis session. Thisequilibration is due to the removal of urea from the blood by thedialyzer at a rate that exceeds the rate at which the urea diffuses fromthe intracellular to the extracellular compartment. These delays arefurther associated with the fact that 70% of the total body water iscontained in organs that receive only 20% of the cardiac output. Thus,the urea may be trapped in relatively poorly perfused tissues, such asskin, muscle and bone, and are cleared of urea much less efficientlythan the highly vascularized organs, such as the liver or lungs.

As indicated in FIG. 1, the blood urea concentration follows a curve ofreduction during dialysis which shows a relatively sharp upward bump atthe time dialysis is terminated. In contrast, tissue urea analysis isindicated as a relatively constant curve and believed to have a smallnegative bump in urea concentration at the time dialysis is terminateddue to continued movement of urea from the intracellular to theextracellular compartment and subsequently to the vascular space. Thisbump is, however, highly attenuated due to the vast difference in volumeof the blood versus the extracellular and intracellular compartments.

If the urea rebound was constant and predictable, it would be relativelyeasy to compensate for such rebound through blood analysis of ureaconcentration. However, the magnitude of urea rebound varies greatlyamong dialysis patients and also varies for the same patient betweendialysis sessions. Further, the amount of rebound may also be affectedby the efficiency of the dialysis unit during any given procedure. Ithas been shown that the average rebound at 30 minutes after completionof dialysis is 17%. However, some patients exhibit a rebound as a highas 45%, which results in a 75% error in calculating the ureaconcentration 30 minutes post-dialysis.

The present invention is based on Applicant's recognition that anaccurate, precise and repeatable tissue spectra in the near-infraredrange contains spectral features which may be used to model andcalculate tissue urea concentration for an individual. The presentinvention is further based on a recognition that proper analysis,utilizing a model built on multiple scans, can identify these featureswhich are not readily apparent in visual analysis of a spectral output.

As previously stated, there are two components to the success of themethod of the present invention. First, the method incorporates anapparatus and technique to accurately and repeatably acquire a tissuespectra. The apparatus is sensitive to slight changes in spectral outputat any given wavelength of input and optimizes the overall opticalthroughput both into and out of the tissue sample. Second, the methodrequires an analysis tool including a calibration model based onmultiple spectral results of known urea concentration which is used tocalculate urea concentration in an unknown sample. Each component of theapparatus and method of the present invention are detailed below.

The present invention utilizes an accurate, repeatable and sensitivemethod for non-invasive measurement of a near-infrared tissue spectra.It is recognized that the sample is a complex matrix of materials withdiffering refractive indices and absorption properties. Further, becausemany constituents are present at very low concentrations, it has beenfound to be imperative to couple light into and out from the tissue inan efficient manner. The method of the present invention incorporates anindex-matching medium, fluid or deformable solid, to improve theefficiency of coupling the light both into and out of the tissue sample.

The present invention utilizes light energy in the near-infrared regionof the optical spectrum as an energy source for analysis. Water is byfar the largest contributor to absorption in tissue in the near-infraredregion because of its concentration, as well as its strong absorptioncoefficient. It has been found that the total absorption spectrum oftissue, therefore, closely resembles the water spectrum. It has beenfurther found that tissue greatly scatters light because there are manyrefractive index discontinuities in a typical tissue sample. Water isperfused through the tissue, with a refractive index of 1.33. Cell wallsand other features of tissue have refractive indices closer to 1.5 to1.6. These refractive index discontinuities give rise to scatter.Although these refractive index discontinuities are frequent, they arealso typically small in magnitude and the scatter generally has a strongdirectionality towards the forward direction.

This forward scatter has been described in terms of anisotropy, which isdefined as the cosine of the average scatter angle. Thus, for completebackwards scatter, meaning that all scatter events would cause a photonto divert its direction of travel by 180 degrees, the anisotropy factoris −1. Likewise, for complete forward scatter, the anisotropy factor is+1. In the near-infrared, tissue has been found to have an anisotropyfactor of around 0.9 to 0.95, which is very forward scattering. Forinstance, an anisotropy factor of 0.9 means that an average photon oflight only scatters through an angle of up to 25 degrees as it passesthrough the sample.

In acquiring a tissue spectra, measurements can be made in at least twodifferent modes. It is recognized that one can measure light transmittedthrough a section of tissue, or one may measure light reflected orremitted from tissue. It has been recognized that transmission is thepreferred method of analysis in spectroscopy because of the forwardscattering of light as it passes through the tissue. However, it isdifficult to find a part of the body which is optically thin enough topass near-infrared light through, especially at the longer wave lengths.Thus, the preferred method for measurement in the present invention isto focus on the reflectance of light from the sample.

Photons reflect and refract at refractive index discontinuities, and solight impinging on tissue immediately has a small reflectance at thetissue surface. This is referred to as specular reflectance. Since thislight does not penetrate into the tissue, it contains little informationabout the tissue constituents. This is especially true in light of thephysiology of skin, which possess an outward layer which is essentiallydead and lacks current information about the patient's physiologicalstate. Thus, reflected light energy containing spectral data for ureaanalysis is believed to be that light which is reflected back to thesurface through refractive index discontinuities deeper within thetissue sample. This reflected light energy is referred to as diffuselyreflected light.

Applicants have found that a large fraction of incident photons areabsorbed in the tissue. Those photons which are available for couplingback out of the tissue are likely diverted in their angular path. Infact, by definition, a photon must change direction in order to exit thetissue in a direction towards the input optic. Applicants, however, havefound that a large problem with detection is associated with therefractive index discontinuity between the average tissue refractiveindex and the refractive index of air outside of the tissue. It has beenfound that this discontinuity acting on incident light leads to arefraction and a small specular reflectance of less than about 5percent. However, on the way out, the discontinuity gives rise to acritical angle phenomenon. Because the photon is traveling from a highrefractive index medium to a lower one, a critical angle exists abovewhich a photon is totally internally reflected and will not escape thetissue sample. This critical angle for photons traveling from tissue toair has been found to be about 46 degrees, which presents a problem. Aphoton normally incident on the tissue surface must deviate through alarge angle to exit. Because of the forward directionality ofscattering, this is difficult for a photon to do, and it is very likelyto make a grazing or high angle incidence with the tissue and airinterface. The grazing incidence photons will not escape because thecritical angle is exceeded.

Applicants have found a solution for the differences in refractive indexassociated with coupling light energy exiting tissue to an analyticalinstrument. The solution is the use of an immersion fluid which has verylow absorptivity in the spectral range of interest, and has a viscositycompatible with good flow and coverage, while having a refractive indexwhich closely matches tissue. In preferred embodiments, theindex-matching fluid is preferably minimally or essentiallynon-absorbing of light energy in the wavelengths selected as relevant tomeasurement of urea concentration. The fluid is thusnon-spectroscopically active at desired wavelengths. However, it isbelieved a minimally absorbing index-matching fluid, for example onethat absorbs less than about 10% of the light energy of relevantwavelengths, could still be utilized. A preferred material is afluorinated, chlorinated hydrocarbon polymer oil manufactured byOccidental Chemical under the tradename FLUOROLUBE. FS5 is a preferredFLUOROLUBE. These oils have a refractive index of about 1.38, arenon-toxic, and Applicants have found that it has a spectral signature inthe near-infrared region which is minimal.

Now referring to FIGS. 2 and 3, partial cross-sectional views of twopreferred embodiments of an apparatus for non-invasively acquiring atissue spectrum are depicted. The depictions in FIGS. 2 and 3 areschematic to depict the concept of utilizing an index-matching medium 22in conjunction with a non-invasive sensor element 11 operativelyconnected to an energy source 16 and a spectrum analyzer 30. Therelative size, shape and detail of physical components are not depicted.

The apparatus depicted in FIG. 2 and the apparatus depicted in FIG. 3generally include three elements, an energy source 16, a sensor element11, and a spectrum analyzer 30. The embodiment of FIG. 2 depicts thesensor element as including an input element 20 and an output element26, which can include a single lens system for both input and outputlight energy. The input element 20 and output element 26 are in contactwith a common skin surface 12 of the selected tissue 10. The alternativeembodiment of FIG. 3 depicts an alternative sensor element 11arrangement, wherein the input element 20 and output element 26 arearranged on opposing surfaces 12, 14 of tissue 10. Both embodimentsfunction to give a measure of the absorption of infrared energy by thetissue 10. However, the embodiment of FIG. 2 is utilized to measure thequantity of light energy which is reflected from the tissue 10 by thecomponents or features therein. In contrast, the embodiment of FIG. 3measures the transmission of light energy through the tissue 10. Ineither embodiment, the absorption at various wavelengths can bedetermined by comparison to the intensity of the light energy from theenergy source 16.

The energy source 16 is preferably a wide band, infrared black bodysource. The optical wavelengths emitted from the energy source 16 arepreferably between 1.0 and 2.5 μm. The energy source 16 is operativelycoupled to a first means for transmitting infrared energy 18 from theenergy source to the input element 20. In preferred embodiments, thisfirst means 18 is simply the transmission of light energy to the inputelement 20 through air by placing the energy source 16 proximate theinput element 20.

The input element 20 of the sensor element 11 is preferably an opticallens which focuses the light energy to a high energy density spot.However, it is understood that other beam focusing means may be utilizedin conjunction with the optical lens to alter the area of illumination.For example, a multiple lens system, tapered fibers, or otherconventional optical beam-shaping devices could be utilized to alter theinput light energy.

In both embodiments depicted in FIGS. 2 and 3, an output sensor 26 isutilized to receive reflected or transmitted light energy from thetissue 10. As described in conjunction with a method of analysis below,the embodiment of FIG. 2 has an output sensor 26 which receivesreflected light energy, while the embodiment of FIG. 3 includes anoutput sensor 26 which receives transmitted light through the tissue 10.As with the input element 20, the output element 26 is preferably anoptical lens. Other optical collection means may be incorporated into anoutput element 26, such as a multiple lens system, tapered fiber, orother beam-collection means to assist in directing the light energy tothe spectrum analyzer 30.

A second means for transmitting infrared energy 28 is operativelyconnected to the output element 26. The light transmitted through thesecond means for transmitting infrared energy 28 is transmitted to thespectrum analyzer 30. In a preferred embodiment, the operativeconnection to the output element includes transmission of the reflectedor transmitted light energy exiting the output element through air tothe spectrum analyzer 30. A mirror or series of mirrors may be utilizedto direct this light energy to the spectrum analyzer. In a preferredembodiment, a specular control device is incorporated to separate thespecular reflected light from diffusely reflected light. Such devicesare disclosed in co-pending and commonly assigned application Ser. No.08/871,366, filed Jun. 9, 1997, and entitled “Diffuse ReflectanceMonitoring Apparatus”, the disclosure of which is incorporated herein byreference.

In practicing the method of the present invention, tissue 10 area isselected as the point of analysis. This area can include the skinsurface 12 on the finger, earlobe, forearm, or any other skin surface.Preferably, the area for sampling includes blood vessels near thesurface, and a relatively smooth, uncalloused surface. A preferredsample location is the underside of the forearm. A quantity of anindex-matching medium 22, whether fluid or deformable solid, is thenplaced on the skin surface 12 in the area to be analyzed to couple thesensor element 11, which includes the input element 20 and the outputelement 26 to the instrument.

In acquiring a spectra of the tissue 10, light energy from the energysource 16 is transmitted through the first means for transmittinginfrared energy 18 into the input element 20. The light energy istransmitted from the input element 20 through the index-matching medium22, to the skin surface 12. The light energy contacting the skin surface12 is differentially absorbed by the various components and analytes,such as the urea of interest, contained below the skin surface 12. In apreferred embodiment, the non-absorbed light energy is reflected back tothe output element 26 upon propagating again through the index-matchingmedium 22. The non-absorbed light energy is transmitted via the secondmeans for transmitting infrared energy 28 to the spectrum analyzer 30.

In the alternative embodiment of FIG. 3, the light energy propagatedthrough the input element 20 and first quantity of index-matching medium22 is differentially absorbed by the tissue 10, while a quantity of thelight energy at various wavelengths is transmitted through the tissue 10to the opposing or second skin surface 14. From the second skin surface14, the non-absorbed light energy is propagated through the secondquantity of index-matching medium 24 to the output element 26 withsubsequent propagation to the spectrum analyzer 30 for producing thetissue spectra.

As previously stated, the index-matching medium 22 of the presentinvention is a key to the improved accuracy and repeatability of themethod described above. The index-matching medium can preferably be afluid composition containing chlorofluorocarbons. The composition canalso be a mixture of chlorofluorocarbons and perfluorocarbons. Apreferred composition includes a chlorotrifluoroethylene polymer. Apreferred composition contains about 80% to about 99.8% by weight ofchlorofluorocarbons. As previously stated, the present inventionutilizes an index-matching fluid to optimize the input and output oflight energy to and from a tissue to be analyzed. In its broadest sense,the index-matching fluid of the present invention can be any fluid whichcreates an improved optical interface over that interface which resultsfrom simply placing the probe of the present invention on a skinsurface. Absent the index-matching fluid of the present invention, thisinterface can include gaps which are air filled and cause detrimentalrefraction of light both going into the tissue and exiting the tissue.Thus, any index-matching fluid having a refractive index closer to thatof the tissue at about 1.38 versus the refractive index of air of about1.0 would provide an improved interface.

Applicants have also recognized that the usefulness of the apparatus ofthe present invention requires that the coupling of the sensor berepeatable and that the results be an accurate reflection of the tissueconstituents of the patient. To this end, Applicants have found that itis preferable for the index-matching fluids of the present invention tocontain diagnostic additives. The diagnostic additives provide anassessment of the quality of the lens to tissue interface and/or anassessment of the instrument's present performance.

The non-invasive measurement of tissue spectra by the present inventionis improved by placing an additive into the index-matching fluid thatallows evaluation of the thickness of the fluid when the tissue isplaced in contact with the instrument. In preferred embodiments, theadditive also provides a calibration of the instrument by including acompound of known high absorption at a specified wavelength of light.Such additives also further assure that the correct index-matching fluidis being utilized for the instrument.

Since an index-matching fluid inherently causes a change of height inthe tissue above the sample probe, the measurement of this height canaid in the overall urea analysis, while allowing a path lengthcorrection to be applied to the spectral measurement as a function ofthe tissue height above the sampler. This can insure a reproducible,consistent height is achieved before commencing the spectral measurementof the tissue, and further allows for the adjustment of the heightbefore commencing the spectral measurement of the tissue. In this way,the user can be certain that spurious results are not achieved due toexcess matching fluid height, insufficient index-matching fluid beingutilized, or some other misplacement of the tissue surface relative tothe analyzer.

Laboratory spectrometers utilize a Fourier Transform (FTIR) system whichincorporates a laser reference signal to establish the wavelengths andguarantees that the instrument is calibrated. However, it is likely,instruments that are affordable for an end user will not use a laser,but rather will be dispersion type instruments such as gratings, CCDarrays and others. With such instruments, it is important to makecertain that calibration is proper prior to each analysis of tissuespectra. To this end, Applicants have found that the addition of anadditive which includes a well-defined spectral feature at a knownwavelength of light can be utilized to assure calibration.

The use of a known spectrally active additive to the index-matchingfluid also insures that the end user is using a correct index-matchingfluid for which the instrument has been calibrated and programmed. Theuse of a different index-matching fluid could result in an error in thenon-invasive tissue spectrum by absorbing light energy in the areas ofinterest for identifying an individual.

To accomplish the above repeatability, accuracy and quality assurance, aspectroscopically active agent is preferably added to the index-matchingfluid. The agent preferably has sharp bands of absorption outside theregion of interest to be measured. For example, in a preferred methodfor urea measurement, the agent would be active outside the range of4200 to 7200 wave numbers. The agent could also be active within thisrange so long as there is no significant overlap with wavelengthsactually used to calculate a tissue urea concentration. The additive canbe manufactured by placing an appropriate functional group onperfluorinated hydrocarbons. The perfluorinated hydrocarbons arespectrally inactive in the region of interest, however, the functionalgroup placed upon the perfluorinated hydrocarbons may be spectrallyactive. Further, these functional groups do not interfere with theanalysis of the blood analyte of interest. Exemplary compounds includeperfluoro-2-butyltetrahydrofuran and perfluorosuccinyl chloride.

In an alternative embodiment, the index-matching fluid and diagnosticadditive can comprise the same fluid which provides both functions. Forexample, perfluoro-2-butyltetrahydrofuran can be utilized as anindex-matching medium which improves the optical interface, and at thesame time includes a functional group which makes the compoundspectrographically active in a desired range for diagnostic purposes.

In practicing the present invention, the tissue spectra is determined bymeasuring the light intensity received by the output sensor at thevarious wavelengths which give indications of the absorption at suchwavelengths of the infrared energy as a function of the composition ofthe tissue sample. As is well known in the art, a spectrum analyzer 30of the present invention is able to convert the intensity of theinfrared energy incident on the detector into a proportional amplitudeof voltage. In this way, an output spectrum is defined for the tissueunder analysis. Experimental results documenting the improvementsassociated with the above-identified method for obtaining a tissuespectra are documented in FIG. 4. The top trace, labeled 50, shows theresult obtained when sampling in the previously described mode in theabsence of an index-matching medium. In the bottom trace, labeled 52,100 microliters of chlorotrifuoroethylene polymer was applied to thesurface of the input and output device prior to placing the arm. First,each of the lines drawn, 50 and 52, are each comprised of multiplespectra. With the index-matching fluid, all of the spectra overlay eachother quite closely. This is a good indication that the interface isquite stable. Without the index-matching medium, the interface isextremely unstable and it is clear that the data at a particularwavelength would not be particularly accurate when dealing with smallchanges in concentration of specific constituents that would beindicative of an individual's identity.

Once accurate and repeatable spectral data for tissue analysis isacquired, the second key element of the present invention is themethodology for calibrating the device or instrument to identifyspectral features or combinations of features that can be utilized topredict tissue urea concentration.

In building the model for urea analysis of the present invention, acomputer is utilized which includes a memory having stored therein amultivariate calibration model empirically relating the known ureaconcentration in a plurality of calibration samples to the measuredintensity variations from the calibration samples. The comparisons areconducted at several wavelengths which are defined as having detectedintensity variations in response to variations in urea concentration.Such model is constructed using techniques known by statisticians.

The computer predicts the urea concentration of the tissue sample byutilizing the measured intensity variations, the calibration model, anda multivariant algorithm. Preferably, the computation is made by thepartial least squared techniques as disclosed by Robinson et al. in U.S.Pat. No. 4,975,581, incorporated herein by reference.

It has been found that considerable improvement in detection precisionis obtained by simultaneously utilizing at least several wavelengthsfrom the entire spectral frequency range of the energy source to derivedata for a multi-variant analysis. The multi-variant method allows bothdetection and compensation for interferences, the detection ofmeaningless results, as well as for modeling many types ofnon-linearities. Since the calibration sample used to derive the modelshave been analyzed on a multi-variant basis, the presence of unknownbiological materials in the urea containing tissue does not prevent ordistort the analysis. This is because these unknown biological materialsare also present in the calibration samples used to form the model.Thus, it is important to the model of the present invention that thesamples used to build the model and calibrate the urea analysis areactually samples of living tissue containing other constituents whichwill be present in varying quantities of any future analysis.

Experimental Results

A series of experiments were conducted to determine the feasibility andcapabilities of the present disclosed method and apparatus for opticalmeasurement of the tissue urea concentration. The measurements were allmade non-invasively on the skin, sampled in reflectance from theunderside of a patient's forearm. The spectrometer utilized was a FTIRspectrometer having a 16 cm⁻¹ resolution. The exact spectrometer was aNicolet Magna near-infrared spectrometer. The methodology utilized isthat disclosed previously in the present application. These data wereacquired using an FTIR spectrometer in the wavelength region from 4000cm¹ to 8000 cm¹. Spectroscopic data was obtained from the underside ofthe arm and a one minute sample collection period was used.

The experiment included two patients undergoing dialysis therapy. Acomputer-based calibration model was built for these patients using thepartial least squares technique and was subsequently applied duringtheir dialysis treatment. The calibration model was used to generatenon-invasive prediction results during dialysis therapy. FIGS. 5 and 6show the reduction in blood urea concentration as a function of time.Also shown is the reduction in tissue urea concentration as predicted bythe calibration model from spectral data acquired during dialysis. FIGS.7 and 8 show the delay between the blood and the tissue urea levels. Asthe urea is removed from the blood space, the urea must transfer fromthe extravascular space to the vascular space. This delay is consistentwith what is anticipated due to the fact that the urea is initiallyremoved from the blood and the tissue urea concentration lags behind theblood concentration by a small amount.

In conducting the above set of experiments and in building thecalibration model, NIR spectra were collected from a given subjectbefore dialysis, during dialysis and after dialysis. The post-dialysisspectra were taken approximately one hour later to ensure equilibriumbetween the tissue and blood urea concentrations. The total number ofspectra collected before and after the dialysis was about 50 spectra.These data represent tissue spectra of the given subject in which BUN isunchanging. In separate experiments, the absorbance spectrum of pure BUNwas obtained from known solutions prepared with varying BUNconcentrations. Random amounts of the absorbance spectrum of pure BUNwere added mathematically to the BUN-constant spectra from the givensubject. This procedure ensured that spectral variation in BUN did notcorrelate with any other variations present in the BUN-constant subjectspectra. The partial least squares multivariate calibration model wasthen built from these BUN-augmented subject spectra using the spectralregion between 4000 and 8000 cm⁻¹. The optimal number of PLS factors wasdetermined to be 12 factors. This calibration model was then used topredict the BUN concentration non-invasively from spectra collected fromthe subject during dialysis. The results of the prediction of BUN fromthe spectra collected during dialysis are shown in FIGS. 5-8.

New characteristics and advantages of the invention covered by thisdocument have been set forth in the foregoing description. It will beunderstood, however, that this disclosure is, in many respects, onlyillustrative. Changes may be made in details, particularly in matters ofshape, size, and arrangement of parts, without exceeding the scope ofthe invention. The scope of the invention is, of course, defined in thelanguage in which the appended claims are expressed.

What is claimed is:
 1. A method for assessing the need for hemodialysis,the progress of a hemodialysis procedure or the adequacy of ahemodialysis treatment, said method comprising the steps of: providing ameans for optical analysis of tissue on a patient, said means providingan output spectrum at multiple wavelengths, wherein said output spectrumhas varying intensities as related to absorption by said tissue;coupling said tissue to said means for optical analysis and acquiringsaid output spectrum before, during or after hemodialysis for assessingthe need for hemodialysis, the progress of a hemodialysis procedure orthe adequacy of the hemodialysis treatment, respectively; and,calculating a tissue urea concentration from a mathematical modelrelating said output spectrum to said tissue urea concentration.
 2. Themethod of claim 1, wherein said means for optical analysis of tissuecomprises an apparatus for measuring infrared absorption, wherein saidapparatus includes an energy source emitting infrared energy at multiplewavelengths, including selected wavelengths relevant to ureaconcentration due to absorption by said urea, operatively connected toan input element, said apparatus further including an output elementoperatively connected to a spectrum analyzer, and wherein said couplingstep comprises coupling said input element and said output element tosaid tissue.
 3. The method of claim 2, wherein said coupling stepcomprises coupling said input element and said output element to saidtissue through an index matching fluid.
 4. The method of claim 1,wherein said calculating step comprises utilizing a model based ondifferential absorption data on plural known tissue samples.
 5. Themethod of claim 4, wherein said calculating step comprises utilizing apartial least squares analysis to calculate urea concentration in saidtissue from said model based on differential absorption data.
 6. Anon-invasive method for measuring the concentration of urea in humantissue comprising the steps of: providing an apparatus for measuringinfrared absorption, said apparatus including an energy source emittinginfrared energy at multiple wavelengths, including selected wavelengthsrelevant to urea concentration due to absorption by said urea,operatively connected to an input element, said apparatus furtherincluding an output element operatively connected to a spectrumanalyzer; providing an index-matching medium, said index-matching mediumhaving minimal absorption of infrared energy at said selectedwavelengths, and disposing a quantity of said medium between said humantissue and said input element and output element to couple said elementsto said urea-containing tissue through said index-matching medium; and,irradiating said tissue through said input element with multiplewavelengths of infrared energy so that there is differential absorptionof at least some of said wavelengths; and, collecting at least a portionof the non-absorbed infrared energy with said output element foranalysis of infrared energy absorption with subsequent calculation oftissue urea concentration utilizing a model relating said infraredenergy absorption to said tissue urea concentration.
 7. The method ofclaim 6, wherein said input element and output element are incorporatedinto a single sensor element, and wherein said irradiating step and saidcollecting step are performed utilizing said single sensor.
 8. Themethod of claim 6, wherein said index-matching medium has a refractiveindex closely matched to that of the tissue being irradiated, andwherein said irradiating step and said collecting step are performedutilizing said medium with said closely matched refractive index.
 9. Themethod of claim 6, wherein said index-matching medium further comprisesa diagnostic additive dispersed therein, and wherein said irradiatingstep and said collecting step are performed utilizing said medium withsaid diagnostic additive.
 10. The method of claim 9, wherein saiddiagnostic additive is a spectrographically active agent showing sharpabsorbance at a wavelength other than said selected wavelengths, andwherein said irradiating step and said collecting step are performedutilizing said medium with said spectrographically active agent.
 11. Themethod of claim 9, wherein said diagnostic additive is selected from thegroup consisting of: perfluoro-2-butyltetrahydrofuran, perfluorosuccinylchloride and mixtures thereof, and wherein said irradiating step andsaid collecting step are performed utilizing said medium with saiddiagnostic additive selected from said group.
 12. The method of claim 6,wherein said selected wavelengths include the range of about 4000 cm⁻¹to about 8000 cm⁻¹, and wherein said irradiating step and saidcollecting step are performed using infrared energy within said range.13. A non-invasive method for measuring the concentration of urea inanalyte-containing human tissue comprising the steps of: providing anapparatus for measuring infrared absorption, said apparatus including anenergy source emitting infrared energy at multiple wavelengths,including selected wavelengths relevant to urea concentration due toabsorption by said urea, operatively connected to an input element, saidapparatus further including an output element operatively connected to aspectrum analyzer; providing an index-matching medium, saidindex-matching medium including a chlorofluorocarbon polymer, anddisposing a quantity of said medium between said human tissue and saidinput element and output element to couple said elements to saidanalyte-containing tissue through said index-matching medium;irradiating said tissue through said input element with multiplewavelengths of infrared energy so that there is differential absorptionof at least some of said wavelengths; and, collecting at least a portionof the non-absorbed infrared energy with said output element foranalysis of infrared energy absorption with subsequent calculation oftissue urea concentration utilizing a model relating said infraredenergy absorption to said tissue urea concentration.
 14. The method ofclaim 13, wherein said selected wavelengths range from about 4000 cm⁻¹to about 8000 cm⁻¹, and wherein said irradiating step and saidcollecting step are performed using infrared energy within said range.15. The method of claim 13, wherein said index-matching medium furthercomprises a diagnostic additive dispersed therein, and wherein saidirradiating step and said collecting step are performed utilizing saidmedium with said diagnostic additive.
 16. The method of claim 15,wherein said diagnostic additive is a spectrographically active agentshowing sharp absorbance at a wavelength other than said selectedwavelengths, and wherein said irradiating step and said collecting stepare performed utilizing said medium with said spectrographically activeagent.
 17. The method of claim 15, wherein said diagnostic additive isselected from the group consisting of: perfluoro-2-butyltetrahydrofuran,perfluorosuccinyl chloride and mixtures thereof, and wherein saidirradiating step and said collecting step are performed utilizing saidmedium with said diagnostic additive selected from said group.
 18. Themethod of claim 13, further comprising the step of calculating aconcentration of said blood analyte in said analyte-containing tissuewith said spectrum analyzer by comparing said differential absorption toa model including differential absorption data on plural knownanalyte-containing tissue samples.
 19. The method of claim 18, whereinsaid subsequent calculation step comprises utilizing a partial leastsquares analysis to compare said differential absorption of saidanalyte-containing tissue to said model based on differential absorptiondata.
 20. The method of claim 13, wherein said input element and saidoutput element include optical lenses, and wherein said coupling stepcomprises coupling said optical lenses to said tissue.
 21. The method ofclaim 13, wherein said tissue comprises a skin surface on an undersideof a forearm of a patient, and wherein said coupling step comprisescoupling said input and output elements to said skin surface.
 22. Themethod of claim 13, wherein said index-matching medium has a refractiveindex of about 1.30 to about 1.40, and wherein said irradiating step andsaid collecting step are performed utilizing said medium with saidrefractive index.